Abstract

The goal of this study was to investigate the impact and molecular mechanism of action of two novel proteasome inhibitors, carfilzomib (also named PR-171) and ONX 0912 (previously named PR-047), in head and neck squamous cell carcinoma (HNSCC) cells. HNSCC is the sixth most common cancer in the United States, and is frequently characterized by resistance to conventional chemotherapy. Previously we have shown that HNSCC cells are highly sensitive to the proteasome inhibitor bortezomib. However, clinical application of bortezomib against solid tumors has been hampered by issues of nonspecificity and adverse cytotoxicities. In the current study we examined the effects of two next generation proteasome inhibitors with improved specificities for the chymotrypsin-like activity of the proteasome. Carfilzomib is a well-tolerated epoxyketone, and ONX 0912 is an orally bioavailable derivative. Carfilzomib and ONX 0912 exhibited potent killing activity against eight different HNSCC cell lines. In 48-hour trypan blue exclusion assays IC50 values for carfilzomib ranged from 18.3-70.4 nM, while IC50 values for ONX 0912 ranged from 58.9-185.7 nM. Carfilzomib and ONX 0912 activated HNSCC apoptosis signaling as demonstrated by flow cytometric analysis of Annexin V/PI staining and immunoblot analysis of caspase-3 activation and PARP cleavage. Treatment with carfilzomib or ONX 0912 also led to substantial alterations in the expression levels of Bcl-2 family members, including downregulation of anti-apoptotic Bcl-2 and upregulation of pro-apoptotic Bik, Bim, and Bax; these effects likely serve to promote cell killing by these compounds. At the same time, treatment also led to dramatic upregulation of anti-apoptotic Mcl-1L, and moderate upregulation of anti-apoptotic Bcl-XL; these effects may act to blunt the killing activities of the compounds. Additional studies revealed that carfilzomib and ONX 0912 promote autophagy in HNSCC cells. In HNSCC cells engineered to express GFP-LC3, treatment led to discrete cytoplasmic puncta characteristic of autophagasome formation. Moreover, carfilzomib and ONX 0912 promoted upregulation of Beclin-1, ATG-5, and LC3-II, indicative of autophagy induction. The induction of autophagy by carfilzomib and ONX 0912 was associated with upregulation of JNK enzymes and phosphorylation of Bcl-2. Taken together, our findings demonstrate that carfilzomib and ONX 0912 activate both apoptosis and autophagy signaling in HNSCC cells. In view of the potent killing activities of these compounds against HNSCC cells, carfilzomib and ONX 0912 may represent viable therapeutic agents in this malignancy. Further studies using HNSCC in vivo models are clearly warranted.